The present invention is generally in the area of monoclonal antibodies useful in immunoassays, and in particular is a monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection, Rockville, Md. on Jan. 7, 1997, designated HB-12254, and a derivatized pyridinoline useful in immunoassays binding assays for peptide linked pyridinoline derived from naturally occurring bone collagen.
It is well established that antibodies are useful in immunoassays when the antibodies are directed against specific epitopes characteristic of a particular disease or metabolic condition. As described in U.S. Pat. No. 4,973,666 to Eyre, an antibody reactive with peptide sequence at the bone collagen hydroxypyridinium crosslinks in the amino-terminal of Type 1 Collagen in human urine can be used to correlate bone resorption, which is indicative of osteoporosis.
Osteoporosis is the most common bone disease in man. Primary osteoporosis, with increased susceptibility to fractures, results from a progressive net loss of skeletal bone mass. It is estimated to affect 15-20 million individuals in the United States. Its basis is an age-dependent imbalance in bone remodeling, i.e., in the rates of synthesis and resorption of bone tissue. About 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured peripheral bones. Twelve to 20% of the hip fractures are fatal because they cause severe trauma and bleeding, and half of the surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $7 billion annually (Barnes, O. M., Science, 236,914 (1987)). Osteoporosis is most common in post-menopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, there are a limited number of methods available for measuring bone resorption rates in patients or normal subjects.
Since bone resorption involves degradation of the mineral and the organic matrix, a specific biochemical marker for newly degraded bone products in body fluids would be the ideal index. Several potential organic indices have been tested. For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein in bone and all other connective tissues, is excreted in urine. Its excretion rate is known to be increased in certain conditions, notably Paget's disease, a metabolic bone disorder in which bone turnover is greatly increased. For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation. Singer, F. R., et al. (1978) In: Metabolic Bone Disease, Vol. II (eds. Avioli, L. V. and Krane, S. M.) pp. 489-575, Academic Press, New York. A urinary, blood or saliva, assay for the whole-body output of degraded bone in 24 hours would be much more useful. Mineral studies (e.g., calcium balance) cannot do this reliably or easily.
The polymers of most generic types of vertebrate collagen require the formation of aldehyde-mediated cross-links for normal function. Collagen aldehydes are derived from a few specific lysine or hydroxylysine side-chains by the action of lysyl oxidase. Various di-, tri- and tetrafunctional cross-linking amino acids are formed by the spontaneous intra- and intermolecular reactions of these aldehydes within the newly formed collagen polymers; the type of cross-linking residue varies specifically with tissue type (see Eyre, D. R. et al. (1984) Ann. Rev. Biochem. 53: 717-748). Two basic pathways of cross-linking can be differentiated for the banded (67 nm repeat) fibrillar collagens, one based on lysine aldehydes, the other on hydroxylysine aldehydes. The lysine aldehyde pathway dominates in adult skin, cornea, sclera, and rat tail tendon and also frequently occurs in other soft connective tissues. The hydroxylysine aldehyde pathway dominates in bone, cartilage, ligament, most tendons and most internal connective tissues of the body, Eyre, D. R. et al. (1974) vide supra. The operating pathway is governed by whether lysine residues are hydroxylated in the telopeptide sites where aldehyde residues will later be formed by lysyl oxidase (Barnes, M. J. et al. (1974) Biochem. J. 139, 461). In both pathways and in most tissues the intermediate borohydride-reducible cross-linking residues disappear as the newly formed collagen matures, suggesting that they are relatively short-lived intermediates (Bailey, A. J. et al. (1971) FEBS Lett. 16, 86). Exceptions are bone and dentin, where the reducible residues persist in appreciable concentration throughout life, in part apparently because the rapid mineralization of the newly made collagen fibrils inhibits further spontaneous cross-linking interactions (Eyre, D. R. (1981) In: The Chemistry and Biology of Mineralized Connective Tissues (Veis, A. ed.) pp. 51-55, Elsevier, N.Y., and Walters, C. et al. (1983) Calc. Tiss. Intl. 35: 401-405).
Two chemical forms of 3-hydroxypyridinium cross-link have been identified. Both compounds are naturally fluorescent, with the same characteristic excitation and emission spectra (Fujimoto, D. et al. (1977) Biochem. Biophys. Res. Commun. 76, 1124, and Eyre, D. R. (1981) Develop. Biochem. 22, 50). These amino acids can be resolved and assayed directly in tissue hydrolysates with good sensitivity using reverse phase HPLC and fluorescence detection. (Eyre, D. R. et al. (1984) Analyt. Biochem. 137: 380-388).
In growing animals it has been reported that these mature cross-links may be concentrated more in an unmineralized fraction of bone collagen than in the mineralized collagen (Banes, A. J., et al. (1983) Biochem. Biophys. Res. Commun. 113, 1975). However, other studies on young bovine or adult human bone do not support this concept, (Eyre, D. R. (1985) In: The Chemistry and Biology of Mineralized Tissues (Butler, W. T. ed.) p. 105, Ebsco Media Inc., Birmingham, Ala.).
The presence of collagen hydroxypyridinium cross-links in human urine was first reported by Gunja-Smith and Boucek (Gunja-Smith, Z. and Boucek, R. J. (1981) Biochem J. 197: 759-762) using lengthy isolation procedures for peptides and conventional amino acid analysis. At that time, they were aware only of the hydroxylysyl pyridinoline form of the cross-link. Robins (Robins, S. P. (1982) Biochem J. 207: 617-620) has reported an enzyme-linked immunoassay to measure hydroxylysyl pyridinoline in urine, having raised polyclonal antibodies to this free amino acid conjugated to bovine serum albumin. This assay is intended to provide an index for monitoring increased joint destruction that occurs with arthritic diseases and is based, according to Robins, on the finding that pyridinoline is much more prevalent in cartilage than in bone collagen. In more recent work involving enzyme-linked immunoassay, Robins reports that lysyl pyridinoline is unreactive toward antiserum to hydroxylysyl pyridinoline covalently linked to bovine serum albumin (Robins et al. (1986) Ann. Rheum. Diseases 45, 969-973). Robins' urinary index for cartilage destruction is based on the discovery that hydroxylysyl pyridinoline, derived primarily from cartilage, is found in urine at concentrations proportional to the rate of joint cartilage resorption.
A method for determining the absolute rate of bone resorption by quantitating the concentration of peptide fragments attached to 3-hydroxypyridinium cross-links derived from bone collagen resorption in a body fluid is described by Eyre in U.S. Pat. No. 4,973,666. A preferred antibody for use in these assays is a monoclonal antibody referred to as 1H11, deposited with the American Type Culture Collection, Rockville, Md. under accession number HB10611, as described in U.S. Pat. No. 5,320,970 to Eyre and marketed by Ostex which antibody recognizes specific linear sequences occurring at cross linking sites.
Pyridinoline is a rigid ring structure. Pyridinoline exists in only two forms, hydroxy lysyl pyridinoline and lysyl pyridinoline, as opposed to the myriad of crosslinks and aging that affects the structure and conformation of adjacent peptide sequences. Much of the pyridinoline in urine is a byproduct of cartilage destruction, so that total pyridinoline is not a specific marker for bone resorption. Since pyridinoline is derived from many sources besides bone, it has limited specificity for detecting bone resorption and its reversal.
Other antibodies are being commercially marketed for use in diagnostic assays for osteoporosis, including antibodies marketed by Osteometer (Crosslaps.TM.) and Metra Biosystems (Pyrilinks.TM.) and Pyrilinks D.TM.)). An antibody that could detect mature (as defined by presence of crosslinks) bone collagen fragments, eliminating the dependence on conformation of a peptide sequence, would greatly assist in use of bone specific crosslinker as a marker. Since 1H11 reacts with peptides linked through pyridinoline and not with pyridinoline itself or the linear peptide, i.e., recognition of the peptide is conformationally dependent, the immune reaction is expected to be influenced by factors influencing patient to patient peptide conformation, e.g., peptide sequence variation, isomeric differences in peptide due to crosslinking or other aging changes such as transversion, glycation which can affect conformation, as well as assay conditions affecting conformation.
The antibody marketed by Crosslaps reacts with a linear carboxy-terminal peptide from the alpha-1 chain of human type I collagen. The peptide alpha sequence is EKAHDGGR. This antibody is subject to much the same limitations as described for 1H11.
The Metra Pyrilinks 1 kit provides antibody that reacts with pyridinium crosslinks derived from collagen, including that from type I bone. Both crosslinks, pyridinoline (hydroxylysyl pyridinoline) and deoxypyridinoline (lysyl pyridinoline) are formed by enzyme lysyl oxidase. The Metra Pyrilinks D kit provides antibody that reacts with deoxypyridinoline which has been shown to be more specific to bone than hydroxypyridinoline.
These antibodies differ in their ability to differentiate peptides from different patient populations and body fluids.
It is an object of the present invention to provide a specific antibody for use in a diagnostic assay for osteoporosis using bodily fluids from postmenopausal (PM) women which correlates with bone loss.
It is another object of the present invention to provide pyridinoline analogs for use in immunoassays for pyridinoline.